RNA viruses cause many diseases in wildlife, domestic animals and humans. These viruses are genetically and antigenically diverse, exhibiting broad tissue tropisms and a wide pathogenic potential. The incubation periods of some of the most pathogenic viruses, e.g. the caliciviruses, are very short. Viral replication and expression of virulence factors may overwhelm early defense mechanisms (Xu, 1991) and cause acute and severe symptoms.
There are no specific treatment regimes for many viral infections. The infection may be serotype specific and natural immunity is often brief or absent (Murray et al., 1998). Immunization against these virulent viruses is impractical because of the diverse serotypes. RNA virus replicative processes lack effective genetic repair mechanisms, and current estimates of RNA virus replicative error rates are such that each genomic replication can be expected to produce one to ten errors, thus generating a high number of variants (Hollan, 1993). Often, the serotypes show no cross protection, such that infection with any one serotype does not protect against infection with another. For example, vaccines against the vesivirus genus of the caliciviruses would have to provide protection against over 40 different neutralizing serotypes (Smith et al., 1998a), and vaccines for the other genera of the Caliciviridae are expected to have the same limitations.
Antisense agents have been proposed for treating various types of viral infection. In general, the specific proposals to date can be classified according to the type of virus targeted, the viral-genome target, and the type of oligonucleotide backbone employed in the antisense compound. Among the viruses that have been targeted are vesicular stomatitis virus (Robbins and Lebleu, 1999), influenza virus (Mizuta et al., 1999), hepatitis B virus (Wu and Wu, 1992), human papilloma virus (Alvarez-Salas et al, 1999), herpes simplex virus (Aurelian and Smith, 2000), HIV (Kusunoki et al., Wei et al, 2000) and foot-and-mouth disease virus (Gutierrez et al, 1993). Viral genome targets that have been proposed include the IE-2 gene of cytomegalovirus (Green et al, 2000), a stem-loop structure at the 5′ non-coding region, the translation initiation codon, a core protein coding sequence of the hepatitis C virus, and the second functional initiator AUG of the foot-and-mouth disease virus (Hanecak et al, 1996; Alt et al, 1995; Gutierrez et al, 1993). Finally, a wide variety of antisense backbone structures have been proposed, including the negatively charged phosphorothioate (PSO) backbone oligomers, particularly the phosphorothioate oligodeoxynucleotides (Hanecak et al, 1996; Alt et al, 1995; Gutierrez et al, 1993) and uniformly modified 2′-methoxyethoxy phosphodiester oligonucleotide (Hanecak et al, 1996).
Discovery and development generally involves demonstration of antiviral activity in cell culture. A compilation of antiviral experiments in cell culture is provided in Table 1 below.
TABLE 1In vitro Antiviral Antisense StudiesVirusReferenceHerpesGao et al. (1989) J. Biol. Chem. 264: 11,521SimplexHerpesHoke et al. (1991) Nucl. Acids Res. 19: 5743SimplexHerpesSmith et al. (1986) Proc. Natl. Acad Sci 83: 2787Simplex 1HIV-tatStevenson & Iversen (1989) J. Gen. Virol. 70: 2673HIV-aptamerMatsukura et al. (1987) Proc. Natl. Acad Sci 84: 7706HIV-revMatsukura et al. (1989) Proc. Natl. Acad Sci 86: 4244HIV-gagAgrawal et al. (1989) Proc. Natl. Acad Sci 86: 7790HIV-LTRVickers et al. (1991) Nucl. Acids Res. 19: 3359TARelementVSVAgris et al. (1986) Biochemistry 25: 6268VSV-NLamaitre et al. (1987) Proc. Natl. Acad Sci 84: 1987proteinHPV-E2Cowsert et al. (1993) Antimic. Agent Chemo. 37:HBVGoodarzi et al. (1990) J. Gen Virol. 71: 3021surfacegeneHBVWu & Wu (1992) J Biol Chem 267: 12,436-12,439SV40Graessmann et al. (1991) Nucl. Acids Res. 19: 53InfluenzaKabanov et al. (1990) FEBS Lett. 259: 327InfluenzaLeiter et al. (1990) Proc. Natl. Acad Sci 87: 3430RousZamecnik & Stephenson (1978) Proc. Natl. Acad SciSarcoma75: 280VirusCMV immed.Anderson et al. (1996) Antimic. Agent Chemo. 40: 2004early RNA
Clinical trials have been initiated for antisense therapeutics targeting HIV, HPV, CMV and HCV (Table 2 below), all using phosphorothioate-linked oligonucleotides. As seen, the clinical trial experience to date indicates some failures, although antisense against CMV infection (ISIS2922) has been approved by the FDA, making this the only antisense agent approved by the FDA to date.
TABLE 2Clinical Trials with Antisense for Antiviral TherapyNameCompanyVirusStatusGEM91HybridonHIV-gag250 pts. Discont. 1997ISIS2105ISISHPV (6&11)400 pts. Fail phase IIIISIS2922ISISCMV-IE2HIV retinitis approvedGEM132HybridonCMVPhase IISIS14803ISISHCVPhase I
The initial optimism towards antisense approaches to effective antiviral therapeutics has been blunted. Many of the effective antisense strategies employed in cell culture models (e.g. those in Table 1) have not successfully proceeded to clinical trials. The slow progress is due in part to the lack of robust cell culture models. For example, the HIV isolates that infect cultured cells do not generally reflect those found in the infected population, and the cell culture models do not integrate the roles of the multiple cell types infected. This problem is compounded by the lack of appropriate pre-clinical animal models for the full exploitation of viral gene expression and replication in vivo. Again, in the case of HIV, the human virus either does not infect animals, or, when primates are infected, they do not develop pathology similar to that seen in humans. The risk in developing antisense antiviral agents without robust culture models and appropriate animal models is great.
Thus, there remains a need for an effective antiviral therapy in several virus families, including small, single-stranded, positive-sense RNA viruses in the picornavirus, calicivirus, togavirus and flavivirus families. To meet this need, an antisense agent must be substantially stable against nuclease degradation, able to be taken up readily by virus-infected host cells following compound administration, and targeted against an effective region of the viral genome, that is, able to shut down viral replication.